Immunochemical methods have proven useful for the diagnosis of disease, especially where physical symptoms are ambiguous, asymptomatic carrier states exist, and where the causative agent is difficult to detect by conventional means. These methods have been most commonly used in the past in the examination of serum, lymph and cerebro-spinal fluid.
In the case of disease of the intestinal tract, immunochemical methods suitable for analysis of feces would be potentially useful. By way of example, such usefulness is especially apparent in diseases such as amoebiasis (amoebic dysentery), and giardiasis.
Amoebiasis is an infection of the bowel caused by the protozoan, Entamoeba histolytica. Its world-wide incidence, estimated as high as 50%, makes it a significant world health problem, especially in the under-developed countries. However, even in the United States, there is an estimated incidence of 3%, or approximately 6,000,000 infected persons.
Symptoms of infection are extremely variable. An infection may be asymptomatic or it may result in mild to severe diarrhea. Severe cases can result in ulceration of the large bowel mucosa, intestinal perforation and hemorrhage. Secondary invasion of other tissues, especially of the liver, occurs in some cases (less than 1%). The disease can be fatal. It is estimated that 90% of those infected by Entamoeba histolytica are asymptomatic carriers of the disease.
Diagnosis of the disease is complicated by the wide variety of symptoms it presents, many of which are common to other pathologic conditions. Methods of diagnosis employed in the prior art include both direct and indirect tests. Direct microscopic identification of the infecting organism in a patient's feces or in swabs or washings obtained from the rectum or sigmoid colon provides the best available positive diagnosis. Indirect diagnosis may be made by demonstrating the presence of antibodies against Entamoeba histolytica in the bloodstream.
In the direct method, a positive diagnosis can be made if the organism is identified in microscopic examination of the stool sample. Difficulties of three sorts are encountered with this method. First, the organism, in identifiable form, is often absent from the stools, even when the patient is suffering active symptoms. The reasons why the organism is only occasionally present are not fully understood, but relate in part to the organism's life cycle.
Entamoeba histolytica undergoes a complex life cycle in which it exists in morpholigically distinct forms. The identifiable form is termed a trophozoite, which is characterized by its appearance as a typical amoeboid cell, having a diameter of 10 to 60 microns, an irregular outline, and that is capable of movement by extensions of the cell body. Certain distinguishing cytological characteristics are also present which enable a trained technician to differentiate the organism from non-pathogenic amoebae. The organism may also exist in the form of a cyst and a precystic form. Cysts of Entamoeba histolytica are difficult to distinguish from those of non-pathogenic amoebae which sometimes inhabit the human gut, such as Entamoeba coli or Entamoeba hartmanii, in unstained specimens. A variety of environmental factors within the intestinal tract appears to contribute to the highly sporadic appearance of Entamoeba histolytica. Continuous examination of samples collected over a 6-day period may be required to obtain a sample having intact trophozoites.
A second difficulty with direct microscopic examination is due to the fact that trophozoites of Entamoeba hartmanii are cytologically distinguishable from Entamoeba histolytica only on the basis of size. Cells less than 10 microns in diameter are arbitrarily classified as Entamoeba hartmanii although some overlap in the size ranges of the two organisms may occur. Indeed, opinion is divided as to whether Entamoeba hartmanii and Entamoeba histolytica are in fact distinct species, although the appearance of Entamoeba hartmanii is generally associated with asymptomatic infections. The non-pathogenic Entamoeba coli is also difficult to distinguish microscopically from Entamoeba histolytica.
The third type of difficulty encountered is inherent in the nature of the material sampled. Interference may be encountered due to the presence of particulate matter in the feces, cell debris, clumps of bacteria, white blood cells, oil droplets and kaolin particles from medication taken to control diarrhea. Various techniques are available to remove such interfering materials, but always at the cost of increasing the duration and complexity of the diagnostic procedure. Furthermore, the fecal sample must be examined while fresh and still warm since the trophozoites round up and lose their characteristic morphology as the specimen cools.
Direct microscopic examination of stained specimens may also be employed. This procedure has the advantage that morphological characteristics are easier to see, which aids in the differentiation of Entamoeba histolytica from non-pathogenic amoebae and interfering particles. Stained slides may be several days in preparation and reading, however, and the requirement for samples taken on successive days is not avoided.
Direct microscopic examination of stained or unstained preparations must be carried out by a trained microscopist. The microscopist observes the specimen through a microscope while moving the slide slowly in order to bring all areas of the specimen into the field of view. Intense concentration is demanded. Fatigue drastically reduces efficiency and accuracy of observation after about an hour's viewing. The number of samples which can be processed by a microscopist in a given day is therefore limited. For background, see Brown, H. W., Basic Clinical Parasitology, Chap. 3, pp 18-37, (1975), and Katz, M., in Postgraduate Medicine, Vol. 58, p. 149, (1975).